DNA sequencing is an essential tool in molecular genetic analysis. The ability to determine DNA nucleotide sequences has become increasingly important as an integral component of many medical diagnostics. Historically, the two most commonly used methods for DNA sequencing were the enzymatic chain-termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert. Both methods rely on gel electrophoresis to resolve, according to their size, DNA fragments produced from a larger DNA segment. Since the electrophoresis step as well as the subsequent detection of the separated DNA-fragments were cumbersome procedures, many efforts had been made to develop more efficient sequencing methods, for example, by developing novel technologies that do not use electrophoresis. Research efforts have produced several such techniques including, e.g., sequencing using scanning tunnel electron microscopy (see, e.g., Driscoll et al., Nature 346: 294-96 (1990)), sequencing by hybridization (see e.g., Bains et al., J. Theo. Biol. 135: 308-07 (1988)), and single molecule detection (Jeff et al., Biomol. Struct. Dynamics 7: 301-06 (1989)), to overcome the disadvantages of gel electrophoresis.
In addition, some efforts focused on methods of sequencing based on the concept of detecting the inorganic pyrophosphate (PP) that is released during a DNA polymerase reaction (e.g., as described in WO 93/23564 and WO 89/09283; see Seo et al. “Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides,” PNAS 102: 5926-59 (2005); Hyman, “New method of sequencing DNA” Anal. Biochem. 174: 423-36 (1988)). In these “sequencing by synthesis” methods, as each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, the released pyrophosphate molecule is detected. It has been found that pyrophosphate released under these conditions can be detected enzymatically, e.g., in some applications by the generation of light in the luciferase-luciferin reaction. Such methods allow a user to sequence DNA simply and rapidly whilst avoiding the need for electrophoresis and the use of harmful radiolabels. In addition, these methods have found use in identifying single target bases, e.g., in the mapping of single nucleotide polymorphisms.
One of the first sequencing by synthesis methods was “Pyrosequencing™”, which was developed at the Royal Institute of Technology in Stockholm (see Nyren, “Method for sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation”, U.S. Pat. No. 6,258,568 (2001); WO 98/28440; Ronaghi, et al. Science 281: 363 (1998); Alderbom et al., (2000), each incorporated herein by reference in their entireties for all purposes). The method, in contrast to conventional Sanger sequencing, adds nucleotides one by one during the sequencing reaction. In some implementations the principle is as follows: A single stranded DNA fragment (attached to a solid support), carrying an annealed sequencing primer acts as a template for the reaction. In the first two dispensations, substrate and enzyme mixes are added to the template. The enzyme mix consists of four different enzymes; DNA polymerase, ATP-sulfurylase, luciferase and apyrase. The nucleotides are sequentially added one by one according to a specified order dependent on the template and determined by the user. If the added nucleotide matches the template, the DNA polymerase incorporates it into the growing DNA strand and PPi is released. The ATP-sulfurylase converts the PPi into ATP, and the third enzyme, luciferase, transforms the ATP into a light signal. Following these reactions, the fourth enzyme, apyrase, degrades the excess nucleotides and ATPs, and the template is ready for the next reaction cycle, i.e. another nucleotide addition. Since no PPi is released unless a nucleotide is incorporated, a light signal is produced only when the correct nucleotide is incorporated.
In a related method, the incorporation of a nucleotide during sequencing-by-synthesis is detected by a change in the heat or pH of the reaction solution (see, e.g., U.S. Pat. No. 7,932,034). In one implementation of these methods, a template strand having an attached primer is immobilized in a small volume reaction mixture, with the reaction mixture in contact with a sensitive calorimeter, which detects the heat of reaction from incorporation of a complementary base (dNTP) in the presence of appropriate reagents (DNA polymerase, and polymerase reaction buffer). Alternatively, a pH meter may be used to measure changes in pH resulting from the reaction. The bead will have template DNA attached to it, where the sequence of the template DNA molecule is the same in each of numerous strands attached to the bead, e.g., through biotin. In a known protocol, for example, 5 pg of immobilized template DNA is used. The template DNA is prepared with a known segment for attachment of a primer. In some applications, calorimetric detection is the preferred detection scheme because it allows for more sensitive detection than pH-based schemes.
In pH-based methods, pH monitoring is often performed by use of a microcantilever or a field-effect transistor (FET) sensitive to hydrogen ion concentration. In the microcantilever devices, a pH sensor with ultrahigh sensitivity was developed based on a microcantilever structure with a lithographically defined crosslinked copolymeric hydrogel. Silicon-on-insulator wafers were used to fabricate cantilevers on which a polymer consisting of poly (methacrylic acid) (PMAA) with polyethylene glycoldimethacrylate was patterned using free-radical UV polymerization. As the pH around the cantilever was increased above the pKa of PMAA, the polymer network expanded and resulted in a reversible change in surface stress causing the microcantilever to bend. These devices have a sensitivity reported to be 5×10−4 pH.
In the FET devices, a chemical-sensitive FET, or more particularly an ion-sensitive FET (ISFET), is used to facilitate measurement of the hydrogen ion concentration of a solution. An ISFET is an impedance transformation device that is fabricated using conventional complementary metal oxide semiconductor (CMOS) technology, operates in a manner similar to that of a metal oxide semiconductor field effect transistor (MOSFET), and is particularly configured to selectively measure ion activity in a solution (e.g., hydrogen ion). Examples of these devices are provided, e.g., in U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073, and 20100137143.
Other commercially available pH meters can measure pH changes as low as 0.001. These meters contain several inputs for indicator (e.g., ion-sensitive, redox), reference electrodes, and temperature sensors such as thermoresistors or a thermocouple. The electronic pH meters typically use potentiometric methods, that is, one measures a potential difference between known reference electrode and the measuring pH electrode.
However, the sequencing methods mentioned above are not without drawbacks. For example, many methods rely on relatively sophisticated detection schemes that rely on, for example, chemiluminescence or a FET to detect the release of pyrophosphate or pyrophosphate analogues. Chemiluminescence is detected by photon counting devices and is associated with light-tight detection methods. Field effect transistors remain fairly sophisticated to fabricate and are subject to “salt effects” (e.g., Debye effects) that can inhibit the sensitivity of detection. Consequently, a need remains for a pragmatic and reliable technology for monitoring base polymerization during a sequencing by synthesis reaction.